Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract

The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.     

Effect of Palmitic and Lauric Acids on the Phospholipid Bilayer Membrane Conductivity

Palmitic acid increased the conductivity of BLM from mitochondrial phospholipids when they were dissolved in a mixture of decane and chlorodecane, and was ineffective when phospholipids were dissolved in decane. Lauric acid produced an increase in the membrane conductivity independently of the phospholipid type in the membrane-forming solutions (mitochondrial phospholipids, asolectin, lecithin with cholesterol) and their solvents (decane or decane with chlorodecane). The results show that discrepancies between published data concerning fatty acid effects on the BLM conductivity may be explained by differences in phospholipids, their solvents and fatty acid used.     

Examining the Origins of the Hydration Force Between Lipid Bilayers Using All-Atom Simulations

Using 237 all-atom double bilayer simulations, we examined the thermodynamic and structural changes that occur as a phosphatidylcholine lipid bilayer stack is dehydrated. The simulated system represents a micropatch of lipid multilayer systems that are studied experimentally using surface force apparatus, atomic force microscopy and osmotic pressure studies. In these experiments, the hydration level of the system is varied, changing the separation between the bilayers, in order to understand the forces that the bilayers feel as they are brought together. These studies have found a curious, strongly repulsive force when the bilayers are very close to each other, which has been termed the “hydration force,” though the origins of this force are not clearly understood. We computationally reproduce this repulsive, relatively free energy change as bilayers come together and make qualitative conclusions as to the enthalpic and entropic origins of the free energy change. This analysis is supported by data showing structural changes in the waters, lipids and salts that have also been seen in experimental work. Increases in solvent ordering as the bilayers are dehydrated are found to be essential in causing the repulsion as the bilayers come together.     

Dissociation of a Dynamic Protein Complex Studied by All-Atom Molecular Simulations

The process of protein complex dissociation remains to be understood at the atomic level of detail. Computers now allow microsecond timescale molecular-dynamics simulations, which make the visualization of such processes possible. Here, we investigated the dissociation process of the EphA2-SHIP2 SAM-SAM domain heterodimer complex using unrestrained all-atom molecular-dynamics simulations. Previous studies on this system have shown that alternate configurations are sampled, that their interconversion can be fast, and that the complex is dynamic by nature. Starting from different NMR-derived structures, mutants were designed to stabilize a subset of configurations by swapping ion pairs across the protein-protein interface. We focused on two mutants, K956D/D1235K and R957D/D1223R, with attenuated binding affinity compared with the wild-type proteins. In contrast to calculations on the wild-type complexes, the majority of simulations of these mutants showed protein dissociation within 2.4 μs. During the separation process, we observed domain rotation and pivoting as well as a translation and simultaneous rolling, typically to alternate and weaker binding interfaces. Several unsuccessful recapturing attempts occurred once the domains were moderately separated. An analysis of protein solvation suggests that the dissociation process correlates with a progressive loss of protein-protein contacts. Furthermore, an evaluation of internal protein dynamics using quasi-harmonic and order parameter analyses indicates that changes in protein internal motions are expected to contribute significantly to the thermodynamics of protein dissociation. Considering protein association as the reverse of the separation process, the initial role of charged/polar interactions is emphasized, followed by changes in protein and solvent dynamics. The trajectories show that protein separation does not follow a single distinct pathway, but suggest that the mechanism of dissociation is common in that it initially involves transitions to surfaces with fewer, less favorable contacts compared with those seen in the fully formed complex.     

Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav1_82–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav1_82–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav1_82–136 insertion depth in the bilayer), corroborate that Cav1_82–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav1_82–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.     

Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav1_82–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav1_82–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav1_82–136 insertion depth in the bilayer), corroborate that Cav1_82–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav1_82–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.     

Molecular Dynamics Simulations of a DMPC Bilayer Using Nonadditive Interaction Models

We present a polarizable force field based on the charge-equilibration formalism for molecular dynamics simulations of phospholipid bilayers. We discuss refinement of headgroup dihedral potential parameters to reproduce ab initio conformational energies of dimethylphosphate calculated at the MP2/cc-pVTZ level of theory. We also address the refinement of electrostatic and Lennard-Jones (van der Waals) parameters to reproduce ab initio polarizabilities and water interaction energies of dimethylphosphate and tetramethylammonium. We present results of molecular dynamics simulations of a solvated dimyristoylphosphatidylcholine bilayer using this polarizable force field as well as the nonpolarizable, fixed-charge CHARMM27 and CHARMM27r force fields for comparison. Calculated atomic and electron-density profiles, deuterium order parameters, and headgroup orientations are found to be consistent with previous simulations and with experiment. Polarizable interaction models for solvent and lipid exhibit greater water penetration into the lipid interior; this is due to the variation of water molecular dipole moment from a bulk value of 2.6 Debye to a value of 1.9 Debye in the membrane interior. The reduction in the electrostatic component of the desolvation free-energy penalty allows for greater water density. The surface dipole potential predicted by the polarizable model is 0.95 V compared to the value of 0.8 V based on nonpolarizable force-field calculations. Effects of inclusion of explicit polarization are discussed in relation to water dipole moment and varying charge distributions. Dielectric permittivity profiles for polarizable and nonpolarizable interactions exhibit subtle differences arising from the nature of the individual component parameterizations; for the polarizable force field, we obtain a bulk dielectric permittivity of 79, whereas the nonpolarizable force field plateaus at 97 (the value for pure TIP3P water). In the membrane interior, both models predict unit permittivities, with the polarizable models contributing from one to two more units due to the optical dielectric (high-frequency dipole fluctuations). This contribution is a step toward the continuing development of a CHARMM (Chemistry at Harvard Molecular Mechanics) polarizable force field for simulations of biomacromolecular systems.     

Calcium Currents Are Enhanced by ��2��-1 Lacking Its Membrane Anchor

The accessory α₂δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α₂ and δ. All α₂δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α₂δ subunits, we have now examined the properties of α₂δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α₂δ-1ΔC-term). We find that the majority of α₂δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α₂δ-1ΔC-term with Ca_V2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α₂δ-1. These results call into question the role of membrane anchoring of α₂δ subunits for calcium current enhancement.     

Facile Lipid Flip-Flop in a Phospholipid Bilayer Induced by Gramicidin A Measured by Sum-Frequency Vibrational Spectroscopy

The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.     

Interaction of an Amphiphilic Peptide with a Phospholipid Bilayer Surface by Molecular Dynamics Simulation Study

Abstract

Corticotropin-releasing factor (CRF) is the principal neuroregulator of adrenocorticotropic hormone (ACTH) secretion. Previous experiments have demonstrated that CRF binds avidly to the surface of single egg phosphatidylcholine vesicles and its amphiphilic secondary structure might play an important role in the function. In this study, the interaction of the residues 13–41 in human CRF with the surface of a DOPC bilayer was investigated by molecular dynamics (MD) simulation in order to understand the role of the membrane surface in the formation of the amphiphilic α helix as well as to determine the effects of the peptide on the lipid bilayer. The model used included 60 DOPC molecules, 1 helical peptide (CRF_13–41) on the bilayer surface, and explicit waters of solvation in the lipid polar head group regions, together with constant-volume periodic boundary conditions in three dimensions. The MD simulation was carried out for 510 ps. In addition, CRF_13–41, initially in a helical form, was simulated in vacuo as a control. The results indicate that while it was completely unstable in vacuo, the peptide helical form was generally maintained on the bilayer surface, but with distortions near the terminal ends. The peptide was confined to the bilayer headgroup/water region, similar to that reported from neutron diffraction measurement of tripeptides bound to the phosphatidylcholine bilayer surface (Ref 1). The amphiphilicity of the peptide matched that of the bilayer headgroup environment, with the hydrophilic side oriented toward water and the hydrophobic side making contact with the bilayer hydrocarbon core. These results support the hypothesis that the amphiphilic environment of a membrane surface is important in the induction of peptide amphiphilic α-helical secondary structure. Two major effects of the peptide on the lipids were found: the first CH₂ segment in the lipid chains was significantly disordered and the lipid headgroup distribution was broadened towards the water region.     

Reconstitution of the Mitochondrial ATP-Dependent Potassium Channel into Bilayer Lipid Membrane1

Electrical properties and regulation of the mitochondrialATP-dependent potassium channel were studied. The channel protein wassolubilized from the mitochondrial membrane using an ethanol/water mixture.Reconstituted into a bilayer lipid membrane BLM), the protein formed aslightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl.Often, several channels worked simultaneously (clusters) when many channelswere incorporated into the BLM. The elementary channel and the clusters wereboth highly potassium selective. At concentrations of 1 to 10 M, ATPfavors channel opening, while channels become closed at 1–3 mM ATP. GDP(0.5 mM) reactivated the ATP-closed channels without affecting the untreatedchannels. The sulfhydryl-reducing agent ditiothreitol increased the openprobability at concentrations of 1 to 3 mM, but damaged the selectivity ofthe channel.     

Building KCNQ1/KCNE1 Channel Models and Probing their Interactions by Molecular-Dynamics Simulations

The slow delayed rectifier (I_Ks) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of I_Ks-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in I_Ks channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.     

Building KCNQ1/KCNE1 Channel Models and Probing their Interactions by Molecular-Dynamics Simulations

The slow delayed rectifier (I_Ks) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of I_Ks-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in I_Ks channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.     

Stabilization of the Bio-membrane by Small Molecules: Interaction of Trehalose with the Phospholipid Bilayer

Abstract

Anhydrobiotic organisms undergo periods of acute dehydration during their life cycle. It is of interest to understand how the biomembrane remains intact through such stress. A di-saccharide, trehalose, which is metabolised during anhydrobiosis is found to prevent disruption of model membrane systems (1). Molecular modelling techniques are used to investigate the possible mode of interaction of trehalose with a model monolayer. The objective is to maximise hydrogen bonding between the two systems. A phospholipid matrix consisting of l,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) is chosen to represent the monolayer. The crystal structure of DMPC (2) reveals that there are two distinct conformers designated as A and B. An expansion of the monolayer, coplanar with its surface, results in the trehalose molecule being accomodated in a pocket formed by four B conformers. One glucose ring of the sugar rests on the hydrophobic patch provided by the choline methyls of an A conformer. Five hydrogen bonds are formed involving the phosphate oxygens of three of the surrounding B conformers. The model will be discussed with reference to relevant experimental data on the interaction.     

结合AFM探测副伤寒沙门氏茵B感染红细胞膜的生物力学特性

采用原子力显微镜和衍射显微术,在纳米精确尺度探测副伤寒沙门氏菌B（Sp B）感染宿主红细胞（RBC）膜微观结构和力学特性,涉及细胞的形变、膜面内剪切模量和弯曲模量。结合这两种单分子测量技术,利用相关的数学模型表述RBC膜对菌体印B的入侵非常敏感。实验结果显示,不同感染期间的SpB寄生菌体,能够引起宿主RBC膜结构改变,形变能力降低,膜剪切模量和弯曲模量显著增加。这些力学特性的变化影响RBC的输氧和循环功能。实验结果表明,印B具有独特的鞭毛调控系统,入侵的毒性菌体寄生蛋白与血影蛋白网络中的运输蛋白有特异结合位点,导致RBC膜骨架网络、波动力学和细胞内、外基质都产生应激反应,这有可能为理解勋曰感染RBC的发病机理和寄生途径提供一些新的实验思路和分析依据。     

Influence of Phospholipid Species on Membrane Fluidity: A Meta-analysis for a Novel Phospholipid Fluidity Index

Generalized membrane lipid composition determinants of fluidity have been widely investigated, including phospholipid/cholesterol ratio and unsaturation index. Individual phospholipids differ in their physical characteristics, including their interaction with cholesterol and level of unsaturation, emphasizing the importance of examining their individual influence on membrane fluidity. Thus, the purpose of this study was to examine the dominant phospholipids of biological membranes (phosphatidylcholine, PC; phosphatidylethanolamine, PE; sphingomyelin, SM) through a meta-analysis to assess the validity of an inclusive phospholipid fluidity index (PFI = PC/(PE + SM)) as a determinant for membrane fluidity (expressed as polarization of fluorescent probe 1,6 diphenyl-1,3,5-hexatriene) in comparison to previous phospholipid ratios (PC/PE and PC/SM). The results demonstrate that all indices significantly predicted membrane fluidity at 25°C (based on 10–13 data points). In contrast, only PFI approached significance when predicting membrane fluidity at 37°C (P = 0.10 based on five points). As a result, PFI appears to be the only phospholipid index close to significantly predicting membrane fluidity at mammalian physiological temperature. Because this meta-analysis only assessed studies using mammalian membranes, future work should experimentally assess the validity of the PFI utilizing membranes from mammals and a variety of other species and tissues at their respective physiological temperatures.     

Membrane Tethering of SepF,a Membrane Anchor for the Mycobacterium tuberculosis Z-ring

Bacterial cell division begins with the formation of the Z-ring via polymerization of FtsZ and the localization of Z-ring beneath the inner membrane through membrane anchors. In Mycobacterium tuberculosis (Mtb), SepF is one such membrane anchor, but our understanding of the underlying mechanism is very limited. Here we used molecular dynamics simulations to characterize how SepF itself, a water-soluble protein, tethers to acidic membranes that mimic the Mtb inner membrane. In addition to an amphipathic helix (residues 1–12) at the N-terminus, membrane binding also occurs through two stretches of positively charged residues (Arg27-Arg37 and Arg95-Arg107) in the long linker preceding the FtsZ-binding core domain (residues 128–218). The additional interactions via the disordered linker stabilize the membrane tethering of SepF, and keep the core domain of SepF and hence the attached Z-ring close to the membrane. The resulting membrane proximity of the Z-ring in turn enables its interactions with and thus recruitment of two membrane proteins, FtsW and CrgA, at the late stage of cell division.     

The Taz1p Transacylase Is Imported and Sorted into the Outer Mitochondrial Membrane via a Membrane Anchor Domain

Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome.